Antifreeze proteins (AFP) are a family of peptides naturally found in fish inhabiting extremely cold polar marine waters and are believed to protect them from freezing. While ordinary fish would freeze solid in the 28.5.degree. F. to 32.degree. F. polar oceans, fish containing AFP stay fluid and flexible in these supercooled waters. The mechanism of action of AFP is still largely unknown. Aqueous solutions containing AFP possess many unusual freezing properties. For example, AFP lowers the freezing point of a solution in a non-colligative manner resulting in little or no effect on the melting point. Hence, an AFP solution may have a freezing point of -2.degree. C., but a melting point of -1.degree. C. In contrast, salts typically depress the freezing point and melting point equally. Along the same lines, AFP lowers the temperature at which an ice crystal will grow but does not lower the melting point. It is this property of AFP that may be the most important in regards to protecting the fish because it is the physical damage caused by ice crystal growth through membranes which is a major cause of freezing injury and death. It has been proposed that the activity of AFP is the result of its ability to inhibit ice growth through adsorption to the ice surface (Raymond, J. A. & DeVries, A. L. 1977 PNAS USA 74, 2589-2593). For example, ice normally grows in directions perpendicular to the c axis (Fletcher, N. H. 1970 "The Chemical Physics of Ice", Cambridge Univ. Press, Cambridge); AFP inhibits growth in these directions (Raymond, J. A. Wilson, P. & DeVries, A. L. 1989 PNAS USA 86, 881-885).
Temperature fluctuations during transport and storage of frozen foods can result in numerous cycles of freeze-thaw. In a product like ice cream, this freeze-thaw causes an increase in ice crystal size and a subsequent degradation in texture and product quality (described as "icy", "grainy", or "loss of homogeneity"). This occurs at temperatures near the product's freezing point where an equilibrium between water in the solid and liquid phases occurs resulting in larger ice crystals growing at the expense of smaller ones. Ice cream manufacturers try to overcome this problem by storage and shipping product at very cold temperatures far below the product's melting point (-20.degree. F.). However, once the product gets to the distributors, the stores, and to the customers' home frost-free freezer (0.degree. F.), the manufacturer no longer has control of the product storage temperature and degradation of quality can occur rapidly.
Ice crystal growth is also a problem in many other frozen food products. For example, the cell structure in frozen baked breads and cakes is broken down and the product dehydrated by ice crystal growth (migration of the small homogeneous ice crystals to large heterogeneous ones) over time resulting in a degradation in texture. Ice crystals also destroy the viability of microbial organisms used in the preparation and processing of various foods. For example, in "live dough" bread and pizza products, the fermentative ability of yeast required to raise a previously frozen dough is reduced or destroyed because ice crystal growth rips apart the organisms' membranes, killing them. Also, live cultures of microorganisms (bacteria and yeast) used in the production of food products, such as yogurt, cheese, sausage, beer, wine, etc., must be continually propagated and maintained at high costs because freezing results in lost viability.
Ice crystals are also a problem in many non-food products. For example, in the biotechnology industry, microbial cell lines including bacteria, fungi, and animal cells (e.g., "monoclonal cell lines") are preserved by freezing, but by conventional methods, only a fraction of the cells survive. In the medical industry, a significant proportion of cells comprising the tissues of organs are destroyed by freezing, thus making frozen organs currently unsuitable for transplantation. In the paint industry, the freezing of latex paint results in the separation of pigments and binders from the solvent (water), destroying the homogeneity of the components, resulting in irreversible gelation. These are only a few of numerous examples of potential applications of antifreeze proteins.
Isolation and characterization of antifreeze proteins from different fish have established the existence of four distinct classes of antifreeze proteins, one class of antifreeze glycoproteins and three classes of antifreeze peptides. The antifreeze glycoproteins, (AFGPs) are naturally found in the Antarctic Nototheniid, Dissostichis mawsoniarei, and are glycotripeptide polymers of Alanine-Alanine-Threonine with a disaccharide linked to the Threonine residue. This series of eight glycopeptides range in size from 2,400 to 34,000 Da (DeVries, A. L. 1983. Annu. Rev. Physiol. 45, 245-260). The other three classes of antifreeze proteins (AFPs) consist only of amino acid chains, are rather diverse and include the alanine-rich AFPs, cysteine-rich AFPs and a third class that is neither rich in alanine nor cysteine (Hew, C. L. Scott, G. K. & Davies, P. L. in "Living in the Cold, Physiological and Biochemical Adaptation". eds Heller, H. D. et al. 117-123 Elsevier, N.Y., 1986; Hew et al. 1984, J. Chromatog. 296:213-219).
The cysteine-rich AFPs are naturally found in the sea raven and are 9,000 to 10,000 Da in size. The class of AFPs which is neither cysteine-rich nor alanine-rich is also the most diverse. The AFPs from the Antarctic and North Atlantic Zoarcids Rhigophila dearborni, Macrozoarce americanus, and Austrolycicthys brachycephalus are similar ranging from 61 to 64 nonrepeating residues, with 56% to 69% homology, and lacks both alpha and beta helical tertiary structure (Raymond, J. A. Wilson, P. & DeVries, A. L. 1989 PNAS USA 86, 881-885). In contrast, the AFP from the Cottid Hemitripteris americanus is 17,000 Da in size, has a non-repeating sequence without significant homology to the zoarcid sequences, and possesses both alpha and beta helical secondary structure (Ng, N. F. et al. 1986, J. Biol. Chem. 261, 15690-15695).
The alanine-rich AFPs are naturally found in the North Atlantic Cottid, Myoxocephalus scorpius, and the winter flounder Pseudopleuronectus americanus. This class of AFPs range in size from 2,900 to 10,000 Da depending on the method of measurement. These peptides have a common motif comprised of an 11 amino acid repeating sequence with high alanine content which is purported to contribute the antifreeze activity (Fourney et al. 1984, Can J. Zool. 62, 38-33). The winter flounder AFPs have been among the most thoroughly studied of the alanine-rich AFPs, and amino sequence analysis of the two most abundant AFP, antifreeze protein component A and antifreeze component B (hereafter referred to as AFP A and AFP B), reveal that they each are comprised of 37 amino acids (containing three 11 amino acid repeats) resulting in a size of 3300 daltons.
The winter flounder, Pseudopleuronectus americanus, produces a class of at least seven closely related alanine-rich antifreeze proteins; however, two of the seven, AFP A and AFP B make up 55% and 35% of the total AFP mass, respectively. Their amino acid sequences have been determined (Duman, J. G. and DeVries, A. L. 1976. Comp. Biochem. Physiol. 54B, 375-380; DeVries, A. L., and Lin, Y. 1977, Biochem. Biophys. Acta. 495, 368-392; and reviewed in Fourney et al. 1984, Can. J. Zool. 62, 28-33) and are taken from Pickett et al. (1984, Eur. J. Biochem. 143, 35-38). The amino acid sequence of AFP A is shown in SEQ ID NO:1. AFP B is identical to AFP A except that residue 18 is an Ala, not a Lys; residue 22 is a Lys, not a Glu; and residue 26 is an Asp, not an Ala.
The winter flounder AFP genes have been cloned and sequenced (Davies et al. 1982, PNAS USA 79, 335-339; Davies, P. L. et al. 1984, J. Biol. Chem. 259, 92419247; Pickett et al. 1984, Eur. J. Biochem. 143, 35-38; Scott, G. K., Hew, C. L., and Davies, P. L., 1985, PNAS USA 82, 2613-2617; Fourney et al. 1984, Can. J. Zool. 62, 28-33). From the DNA sequence information, it was deduced that the AFP gene has an intervening sequence which is removed during DNA maturation and that AFP is synthesized as an 82 amino acid preproprotein. A typical winter flounder AFP gene DNA sequence encoding AFP A is taken from Scott et al. 1988 (J. Mol. Evol. 27, 29-35) and reported at SEQ ID No:2 and at FIGS. 5A-5D.
The promoter (TATAAAA), transcription start site (+1, A), translation termination codon (TAA), and polyadenylation signal (GAATAAA) are underlined in FIGS. 5A-5D. The intervening sequence is written in lower case letters, and the predicted sequence for the gene product is shown above the DNA sequence. The predicted 82 amino acid preproprotein sequence encoded by this gene is identical to antifreeze protein A preprotein isolated from winter flounder (Davies et al. 1982, PNAS USA 79, 335-339).
The genome arrangement of the winter flounder AFP genes have been studied in detail and are found to make up a multigene family of AFP genes, each comprising about 1000 base pairs of DNA tandemly linked and clustered in the genome (Davies et al. 1984, J. Biol. Chem. 259, 9241-9247; Scott et al. 1985, PNAS USA 82, 2613-2617; Scott et al. 1988, J. Mol. Evol. 27, 29-35). Studies in winter flounder (Hew et al. 1978, Biochem. Biophys. Res. Commun. 85,421-427; Pickett, et al. 1983, Biochem. Biophys. Acta 739, 97-104) revealed that an 82-residue preproprotein is synthesized in the liver where it is processed to a 59 residue proprotein which in turn is secreted into the serum. In the serum the 59-residue proprotein is matured to the most abundant species, a 37-residue AFP (i.e., AFP A and B). Fractionation of fish serum using Sephadex G75 chromatography followed by Reversed Phase-HPLC however, resolves seven active AFPs, AFP A and B along with five other less abundant species (Fourney et al. 1983, Can. J. Zool. 62, 28-33). One of the other five active AFPs in a 36-residue version of AFP A lacking the C-terminal (37th amino acid) Arginine residue (Pickett et al. 1984, Eur. J. Biochem. 143, 35-38; Fourney et al. 1984, Can. J. Zool. 62, 28-33;).
One method to determine if a molecule has antifreeze protein activity is to measure the thermal hysteresis of a solution in which AFP has been added (Fourney et al. 1983, Can. J. Zool. 62, 28-33). Thermal hysteresis is defined as the difference between the freezing and melting temperatures of a solution. As stated previously, AFP lowers the freezing point of a solution in a non-colligative manner resulting in little or no effect on the melting point. Hence, an AFP solution may have a freezing point of -2.degree. C., but a melting point of -1.degree. C. In contrast, salts typically depress the freezing point and melting point equally. Another method for assessing AFP efficacy is to monitor the rate of ice crystal growth and morphology microscopically (Raymond et al. 1989, PNAS USA 86, 881-885). Ice crystals formed in aqueous solutions grow rapidly, particularly near the melting point; the addition of low levels of AFP drastically reduces the growth rate resulting in smaller ice crystals. However, the art lacks an efficient way to assay for the presence of AFP.
Due to the novel properties that AFP imparts to solutions and particularly its ability to reduce the rate of ice crystal growth, it may have many applications in food, beverage, and non-food products. As a first step, a plentiful supply of antifreeze protein is required to utilize it as a research tool to investigate its applicability as a product ingredient. Although AFP makes up as much as 1% of the winter flounder serum protein in the mid-winter months, the level drop by 100 fold the rest of the year and it is not possible nor commercially feasible to obtain the large quantities which would be required for a single commercial application, such as the frozen food industry. In fact, AFP has not been commercially tested previously because it has not been practical to obtain large enough quantities from fish. Recombinant DNA technology provides a viable alternative. Recently, Peters et al. (1988, Advances in Gene Technology: Protein Engineering and Production, Proceedings of the 1988 Miami Bio/Technology Winter Symposium, ICSU Short Reports Volume 8) reported the cloning and expression of a winter flounder antifreeze proprotein in E. coli, as a Lac Z-proAFP hybrid peptide. After purification of the hybrid Lac Z-proAFP, the Lac Z portion is removed by digestion with a protease (coagulation factor Xa; Nagai, K. & Thogerson, H. C. 1984, Nature 309, 810-812). In this case, the goal of Peters and coworkers was to obtain experimental quantities of the proAFP sufficient to study the winter flounder AFP processing system.
Yeast is considered to be a better host organism for the production of food ingredients because it is a GRAS (generally regarded as safe) organism and it can be made to express, properly process and secrete some heterologous proteins. The problem is that some proteins cannot be produced in yeast (e.g., some are toxic), and others cannot be properly processed and/or secreted. Each protein must be handled on a case by case basis with the probability of success impossible to predict a priori. Another problem is that yeast and winter flounder have different codon preferences and hence obtaining efficient translation of the natural winter flounder gene in yeast is unlikely. This problem can be overcome by direct chemical synthesis of the AFP gene using codons preferred by yeast. Another problem is that the natural winter flounder genes have intervening sequences and the RNA's they encode are unlikely to be properly processed by yeast; and hence obtaining a mature antifreeze protein via expression of a natural winter flounder gene in yeast is unlikely. Again, chemical gene synthesis can overcome this problem by eliminating the intervening sequence from the yeast AFP gene version. Also, the natural winter flounder gene encodes an 82 amino acid preproprotein which in the flounder undergoes a series of proteolytic processing steps to arrive at the mature 37 amino acid active peptide; if produced intracellularly in yeast, it is unlikely that the preproprotein would be processed properly. One approach is the chemical synthesis of a gene lacking the 45 amino acid prepro region, however this would require a Met-residue at the N-terminus. Alternatively, a hybrid gene could be constructed to encode a hybrid protein which can be produced with a convenient proteolytic cleavage site between the leader protein and the N-terminal Asp-residue. Once isolated, the hybrid protein can be processed in vitro.
Another approach is to direct yeast to secrete AFP. One problem is that the natural 45 amino acid winter flounder leader sequence may not function as a secretion and processing signal in yeast since it does not contain the proper processing signal amino acids (e.g., LysArg, ArgArg, or LysLys) between amino acids 45 and 46 to signal the processing necessary to give the desired maturation product. In addition, yeast have no natural processing enzyme to remove the C-terminal Gly, residue 82, of the nascent peptide. Another approach is to chemically synthesize the AFP gene and combine it with a natural yeast expression, processing and secretion system such as the Saccharomyces cerevisiae mating factor alpha one system, resulting in a hybrid gene which encodes a hybrid protein with the proper yeast processing and secretion signals. This requires that the DNA region encoding the mature protein (e.g., alpha factor) be removed and replaced with DNA encoding the mature AFP. The problem is that simple replacement with a heterologous gene may not result in secretion and/or proper processing.